Ascidians are a fascinating group of filter-feeding marine chordates characterized by
rapid evolution of both sequences and structure of their nuclear and mitochondrial genomes.
Moreover, they include several model organisms used to investigate complex biological
processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic
distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other
available mitogenomes, using a novel approach involving detailed whole-mitogenome
comparisons of conspecific and congeneric pairs. The evolutionary rate was quite
homogeneous at both intra-specific and congeneric level, and the lowest congeneric rates
were found in cryptic (morphologically undistinguishable) and in morphologically very
similar species pairs. Moreover, congeneric nonsynonymous rates (dN) were up to two orders
of magnitude higher than in intra-species pairs. Overall, a clear-cut gap sets apart conspecific
from congeneric pairs. These evolutionary peculiarities allowed easily identifying an
extraordinary intra-specific variability in the model ascidian Botryllus schlosseri, where most
pairs show a dN value between those observed at intra-species and congeneric level, yet
consistently lower than that of the C. intestinalis cryptic species pair. These data suggest
ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably,
these ongoing speciation events were undetectable by the cox1 barcode fragment,
demonstrating that, at low phylogenetic distances, the whole mitogenome has a higher
resolving power than cox1. Our study shows that whole-mitogenome comparative analyses,
performed on a suitable sample of congeneric and intra-species pairs, may allow detecting not
only cryptic species but also ongoing speciation events.

Ascidians are a fascinating group of filter-feeding marine chordates characterized by
rapid evolution of both sequences and structure of their nuclear and mitochondrial genomes.
Moreover, they include several model organisms used to investigate complex biological
processes in chordates. To study the evolutionary dynamics of ascidians at short phylogenetic
distances, we sequenced 13 new mitogenomes and analyzed them, together with 15 other
available mitogenomes, using a novel approach involving detailed whole-mitogenome
comparisons of conspecific and congeneric pairs. The evolutionary rate was quite
homogeneous at both intra-specific and congeneric level, and the lowest congeneric rates
were found in cryptic (morphologically undistinguishable) and in morphologically very
similar species pairs. Moreover, congeneric nonsynonymous rates (dN) were up to two orders
of magnitude higher than in intra-species pairs. Overall, a clear-cut gap sets apart conspecific
from congeneric pairs. These evolutionary peculiarities allowed easily identifying an
extraordinary intra-specific variability in the model ascidian Botryllus schlosseri, where most
pairs show a dN value between those observed at intra-species and congeneric level, yet
consistently lower than that of the C. intestinalis cryptic species pair. These data suggest
ongoing speciation events producing genetically distinct B. schlosseri entities. Remarkably,
these ongoing speciation events were undetectable by the cox1 barcode fragment,
demonstrating that, at low phylogenetic distances, the whole mitogenome has a higher
resolving power than cox1. Our study shows that whole-mitogenome comparative analyses,
performed on a suitable sample of congeneric and intra-species pairs, may allow detecting not
only cryptic species but also ongoing speciation events.